Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome

Abstract

A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.

Figure 2

Construction of MCMV BAC genomes and structural analysis of reconstituted virus genomes. (I) Recombinant BAC genomes were generated by homologous recombination in eukaryotic cells with the recombination plasmids pRP2 (a) and pRP3 (b). pRP2 and pRP3 contain 2.2 and 6.6 kb of flanking homologous sequences (open boxes), the BAC vector (shaded boxes) and the gpt gene (crosshatched boxes) flanked by loxP sites (solid box). The EcoRI restriction maps of the right-terminal end of the MCMV wt genome (Upper) and of the resulting BAC genomes pSM3 (a) and pSM4 (b) (Lower) are shown. New EcoRI fragments of 22.9 (a) and 24.3 kb (b) that result from the fusion of the termini in the BAC plasmids are indicated with arrows (above the genomes). Transfection of the BAC plasmids pSM3 and pSM4 into eukaryotic cells generated the recombinant viruses MC96.73 and MC96.74. The terminal EcoRI fragments of 12.3 (a) and 13.7 kb (b) in the linear genomes of these viruses are marked with arrows (below the genomes). Additional restriction enzyme sites indicated are BamHI (B), HindIII (H), and SfiI (S). (II) Structural analysis of BAC plasmids (a) and of reconstituted virus genomes (b, c). (a) Ethidium bromide stained agarose gel of EcoRI digested BAC plasmids pSM3 and pSM4 isolated from E. coli cultures and of MCMV wt DNA isolated from purified virions. (b) Restriction enzyme analysis of reconstituted virus genomes. DNA isolated from MC96.73 and MC96.74 infected cells and MCMV wt DNA was subjected to EcoRI digestion and separated by electrophoresis on 0.6% agarose gels for 14 h. The EcoRI O (O) and the vector fragments (v) are indicated and the size of additional bands is shown to the left. (c) Separation of the EcoRI fragments shown in b after electrophoresis for 28 h.

Figure 3

Construction of ie1 mutant MM96.01 (a) and structural analysis of the mutated BAC plasmid (b) and of the ie1 mutant genome MM96.01 (c). (a) The HindIII site between the HindIII K and L fragments of the MCMV wt genome (Upper) was removed using the EcoRI-HpaI fragment (cross-hatched region) for mutagenesis. The exon-intron structure of the ie1 and ie3 genes is indicated below and protein coding sequences are depicted as cross-hatched boxes. The mutation resulted in a frame shift after 273 codons and created a new stop codon after additional nine codons (solid box). The open box denotes the part of the ie1 ORF, which is not translated in the mutant virus. (b) Ethidium bromide stained agarose gel of the HindIII digested parental BAC plasmid pSM3 and the mutated BAC plasmid pSM_ie1. (c) HindIII pattern of the genomes of recombinant virus MC96.73 and of ie1 mutant MM96.01. The HindIII K and L fragment and the new 15.2-kb fragment are indicated to the left and the size of some HindIII fragments is shown at the right margin.

Figure 1

Strategy for cloning and mutagenesis of the MCMV genome. (A) Viral DNA and recombination plasmids containing the BAC were transfected into eukaryotic cells to generate a recombinant virus. Circular intermediates of the recombinant virus genome were isolated from infected cells and electroporated into E. coli. (B) Mutagenesis of the MCMV BAC plasmid was performed in E. coli by homologous recombination with a mutant allele (mut). (C) The mutated BAC plasmid was transfected into eukaryotic cells to reconstitute recombinant viruses.

Figure 4

Absence of the ie1 protein pp89 in cells infected with ie1 mutant MM96.01. MEF were either mock-infected, infected with recombinant virus MC96.73 or ie1 mutant MM96.01 in the presence of cycloheximide (50 μg/ml) for 3 h to achieve the selective expression of ie genes (16). After removal of cycloheximide, actinomycin D (5 μg/ml) was added and proteins were labeled with [³⁵S]-methionine (1,200 Ci/mmol) for an additional 3 h. Lysis of cells and immunoprecipitations were performed as described (28) using an antiserum directed to the C terminus of the ie1 protein (a) and an ie1/ie3-specific antipeptide serum (b) directed against N-terminal sequences (16, 28). A long exposure of the autoradiograph in b is shown in c.

Figure 5

Growth of the ie1 mutant MM96.01 and the parental virus MC96.73. MEF were infected with MM96.01 (▴) or MC96.73 (□) at a multiplicity of infection of 1. Supernatants of the infected cells were harvested at the indicated time points and titers of progeny were determined by plaque assay.